Mitigating the risk of antimalarial resistance via covalent dual-subunit inhibition of the Plasmodium proteasome

Summary The Plasmodium falciparum proteasome constitutes a promising antimalarial target, with multiple chemotypes potently and selectively inhibiting parasite proliferation and synergizing with the first-line artemisinin drugs, including against artemisinin-resistant parasites. We compared resistance profiles of vinyl sulfone, epoxyketone, macrocyclic peptide, and asparagine ethylenediamine inhibitors and report that the vinyl sulfones were potent even against mutant parasites resistant to other proteasome inhibitors and did not readily select for resistance, particularly WLL that displays covalent and irreversible binding to the catalytic β2 and β5 proteasome subunits. We also observed instances of collateral hypersensitivity, whereby resistance to one inhibitor could sensitize parasites to distinct chemotypes. Proteasome selectivity was confirmed using CRISPR/Cas9-edited mutant and conditional knockdown parasites. Molecular modeling of proteasome mutations suggested spatial contraction of the β5 P1 binding pocket, compromising compound binding. Dual targeting of P. falciparum proteasome subunits using covalent inhibitors provides a potential strategy for restoring artemisinin activity and combating the spread of drug-resistant malaria.


In brief
Inhibitors of the Plasmodium falciparum proteasome have emerged as leading antimalarial candidates. Herein, Deni and Stokes et al. profile a range of diverse chemotypes and identify the vinyl sulfone WLL as the least susceptible to resistance, likely attributable to its covalent, irreversible binding to the b2 and b5 catalytic subunits.

INTRODUCTION
Plasmodium falciparum malaria is a leading cause of mortality among young children in sub-Saharan Africa, who comprised the vast majority of the estimated 619,000 deaths globally in 2021. 1 Recent reports of clinically confirmed de novo emergence of P. falciparum partial resistance to first-line artemisinin (ART) derivatives in East Africa, following the spread of ART partial resistance throughout Southeast Asia, portend a potential worsening of malaria's impact across the African continent. [2][3][4][5][6] To counter drug-resistant malaria, a particularly promising approach is selectively targeting the P. falciparum proteasome. 7 The proteasome is a multi-subunit proteolytic enzyme complex that plays an integral role in maintaining cellular homeostasis in all eukaryotic organisms, including Plasmodium spp. The proteasome contains a 20S core particle with catalytic activity mediated by the b1, b2, and b5 subunits. Access to this core is predominantly regulated by the coupled 19S regulatory particle. 8 This protein complex controls the removal of proteins specifically tagged by polyubiquitin, including ones that are damaged or that temporally regulate diverse processes, including cell cycle progression. Recent evidence also suggests that the P. falciparum 20S proteasome might be secreted into extracellular vesicles that modulate the mechanical properties of native human red blood cells (RBCs) by remodeling their cytoskeletal network, thereby priming RBCs for parasite invasion. 9 Proteasome inhibitors block the development of multiple stages of the Plasmodium life cycle, including oocysts and sporozoites, and broadly interfere with progression through the liver and blood stages, including gametocytogenesis. [10][11][12][13] Novartis, GlaxoSmithKline, and the University of Dundee have realized the value of targeting parasite proteasomes and have developed clinical candidates that inhibit the activity of kinetoplastid proteasomes. 1,[14][15][16][17][18] These compounds are being developed as therapeutics to treat human African trypanosomiasis, Chagas disease, and leishmaniasis.
Large-scale screening and structure-guided chemical optimization efforts have recently identified highly selective inhibitors of Plasmodium spp. proteasomes. 13,[19][20][21][22][23][24][25][26][27] Further refinements to inhibitor design have been made possible by the elucidation of cryoelectron microscopy (cryo-EM) structures of the P. falciparum 20S proteasome. 21,28 These compounds fall broadly into two classes: covalent inhibitors that form irreversible or slowly reversible bonds with the catalytic threonine in the active sites of the catalytic b subunits, or non-covalent inhibitors that reversibly bind the proteasome to block its proteolytic activity. For covalent inhibitors, potency is based on both the initial binding interactions and the subsequent rate of chemical bond formation. Thus, inhibition is time-dependent, and extended exposure to a compound can help compensate for reduced binding affinity to mutant proteasomes. Of the reported covalent inhibitors, the irreversible vinyl sulfones were previously found to have a minimal risk of resistance in vitro. 21,29 This is a key feature of these inhibitors, given that several advanced antimalarial candidates have selected for moderate to highly resistant parasites in human clinical trials. [30][31][32] For those candidates, high-grade resistance in vitro was correlated with in vivo recrudescence, highlighting the importance of understanding resistance liabilities prior to initiating clinical development. [33][34][35] Several Plasmodium-selective proteasome inhibitors have been shown to synergize with ART in vitro, presumably because these compounds interfere with the parasite's response to cellular damage induced by ART treatment. 21,23,29,[36][37][38] ART acts by forming reactive radical species that can alkylate a broad array of parasite biomolecules, causing proteotoxic stress among other forms of cellular damage, such as impaired redox homeostasis. 39,40 Importantly, mutations in the parasite protein K13 (PfKelch13), which mediate resistance to ART, do not interfere with synergy between proteasome inhibitors and ART, including in vitro with P. falciparum cultured asexual blood-stage parasites and in vivo in Plasmodium berghei-infected mice. 27,29,37 This synergy reinforces the appeal of developing Plasmodium-selective proteasome inhibitors as potential new antimalarial medicines. 8,41 Here we examine a panel of inhibitors that represent the main classes of compounds that are selective for Plasmodium proteasomes. Our study, which includes drug susceptibility assays with mutant parasites, in vitro resistance selections, reverse genetics, and molecular modeling, sheds light on compound specificity and identifies classes less likely to readily succumb to resistance, with dual covalent inhibition of the b2 and b5 active sites appearing particularly favorable. These data will help to inform future drug development efforts targeting the P. falciparum proteasome.

RESULTS
Mutations in the P. falciparum 26S proteasome confer distinct patterns of resistance to different inhibitor classes Compound screens and structure-and function-based inhibitor design have yielded compounds with diverse chemotypes that selectively inhibit the P. falciparum proteasome. 13,21,22,42 Resistance to specific chemotypes can be mediated by point mutations that reside mostly within or at the interfaces of the catalytic b subunits that comprise the main substrate-binding pockets of the parasite proteasome. 13,25,29 To determine the degree to which these mutations mediate resistance to different classes of inhibitors, we profiled chemically diverse compounds ( Figure 1) against a panel of proteasome mutant and wild-type (WT) parasite lines in 72 h dose-response assays. These mutant lines harbor singlenucleotide polymorphisms (SNPs) in the b subunits of the Pf20S proteasome core particle or in the Pf19S proteasome regulatory particle, and were previously generated from in vitro resistance selection studies ( Table 1). The mutant lines were selected from the Southeast Asian lines Dd2 (clone B2), Cam3.II (K13 WT or C580Y mutant), or V1/S (K13 WT or C580Y mutant). Compounds were chosen to include several different classes of proteasome inhibitors with different modes of action, which can be classified as covalent irreversible (vinyl sulfones and epoxyketones), covalent reversible (bortezomib, a boronate), or non-covalent reversible (with no reactive electrophile, represented by two asparagine ethylenediamines [AsnEDAs], the macrocyclic peptide TDI-8304, and two N,C-capped peptides). They also have different proteasome subunit selectivity patterns, with most predominantly inhibiting either b5 or b2, while some vinyl sulfones target both these subunits ( Figure 1). From these dose-response assays, we determined the half-maximal growth inhibitory concentration (IC 50 ) of each compound against asynchronous asexual blood-stage P. falciparum cultures (Figure 1, inset). Bortezomib and epoxomicin are non-selective inhibitors that also bind human proteasomes in addition to their antiplasmodial activity and were included for comparative purposes. 11,13,42 Results for dose-response assays testing the activity of the selected inhibitors against our panel of proteasome WT and mutant lines are represented as a heatmap in Figure 2, which shows the log 10 -fold change (the ratio of the IC 50 of the compound tested against a mutant line divided by its IC 50 against the corresponding WT parental line) for each compound against each mutant line. Mean ± SEM IC 50 values are shown in Figure S1 as bar charts that include asterisks to indicate statistically significant changes, with numerical values and fold shifts provided in Tables S1 and S2, respectively.
We first tested the three vinyl sulfone inhibitors WLL, WLW, and EY 4-78, which are all covalent, irreversible inhibitors of the Plasmodium proteasome. 21,23 WLL and EY 4-78 inhibit both the Pf20S b5 and b2 subunits, whereas WLW primarily inhibits b2 21 (Figure 1). WLL and WLW exhibited relatively small shifts in their IC 50 values when tested against our proteasome mutant lines compared with WT parental controls ( Figure 2 and Table S2). For WLL, we observed 2-to 3-fold higher IC 50 values against the WLL-selected b5 A20S and b6 A117V mutant lines 29 compared with their respective parental lines. Collateral sensitivity was also observed, most prominently in b5 A49S mutant parasites (selected with TDI-4258; Table 1) that yielded 5-fold lower IC 50 values relative to the corresponding WT parasites ( Figure 2). Interestingly, parasites harboring an A20V mutation at the same residue as the WLL-selected A20S mutation became more sensitive to WLL as well. The b5 A20V mutant line was selected using the boronate compound MMV1579506, a covalent reversible inhibitor from Takeda that we previously profiled for resistance ( Figures S2A and S2B). These data suggest that resistance to proteasome inhibitors is highly compound-specific, and that mutations selected with one compound can lead to collateral sensitivity to other inhibitor classes.
For WLW, we observed 4-to 8-fold higher IC 50 values against the WLW-selected b2 mutant lines. Interestingly, WLW showed increased potency (with up to an 8-fold lower IC 50 ) against all lines with mutations in b5, as well as against the b6 A117D mutant line, compared with their respective parental lines (Figures 2 and S1; Tables S1 and S2). Nonetheless, some cross resistance between WLL and WLW was observed, with WLW exhibiting somewhat reduced activity against the WLL-selected b6 A117V and b6 S208L mutant lines. These results imply that, in the case of vinyl sulfones, compounds within the same class can have significantly different resistance profiles depending on their subunit specificities.
Optimization of these first-generation Plasmodium-specific vinyl sulfones resulted in generation of a new lead molecule, EY 4-78 (previously ''inhibitor 28''), with less cross-reactivity toward the human proteasome as well as improved solubility and oral bioavailability. 23 Like WLL, EY 4-78 also inhibits both the b2 and b5 20S subunits. We tested EY 4-78 ( Figure 1) against our panel of mutant parasite lines, including WLL-and WLW-selected mutants. Despite chemical similarities between EY 4-78 and WLL, certain mutations conferred distinct patterns of resistance to the two compounds. For example, the b5 M45I mutation, selected with MPI-12, 25 conferred up to a 5-fold gain of resistance to EY 4-78, but resulted in sensitization of parasites to WLL. Nonetheless, the b2 mutant lines selected with WLW were sensitized by as much as 3-to 5-fold to EY 4-78, similar to WLL (Figures 2, S1A, and S3A; Tables S1 and S2). Carmaphycin B, a naturally derived epoxyketone inhibitor, is known to exert potent and selective activity against the Plasmodium proteasome. 22 We tested four carmaphycin B analogs, J-50, J-71, J-78, and J-80 (Figure 1), which share the same epoxyketone reactive group as the parent compound but show improved pharmacological properties. 44 Like the vinyl sulfones, these compounds are also covalent, irreversible peptide inhibitors. All four epoxyketones are highly specific for b5. Our proteasome mutant lines showed nearly identical resistance profiles for all four epoxyketones (Figures 2, S1C, S1D, S3B, and S3C; Tables S1 and S2). Lines harboring b5 mutations exhibited the highest levels of resistance, exceeding increases observed with the vinyl sulfones, with all four epoxyketones showing 8-to 26-fold higher IC 50 values against parasites expressing the b5 M45I and A20V mutations (selected with MPI-12 and MMV1579506, respectively). These data provide evidence of cross resistance between the epoxyketones and the boronate inhibitors MPI-12 and MMV1579506. The four epoxyketones showed 2-to 12-fold higher IC 50 values against b6 mutant lines selected using the vinyl sulfone WLL or the previously published AsnEDA inhibitor PKS21004. 13 In contrast, most epoxyketones showed lower IC 50 values against WLW-selected b2 mutant lines.
We next tested two AsnEDA compounds, WHZ-04 and TDI-4528 ( Figure 1), which, unlike the epoxyketones or vinyl sulfones, bind the proteasome in a non-covalent, reversible manner. 13,24,27 Interestingly, the same two b5 mutations that conferred the highest degrees of resistance to the epoxyketones, namely A20V and M45I, also caused the most significant increases in WHZ-04 and TDI-4258 IC 50 values. In fact, these boronate-selected A20V and M45I mutations conferred greater resistance to WHZ-04 and TDI-4258 than the b6 A117D mutation that was selected with a different AsnEDA, PKS21004. Parasites harboring a separate mutation at the same residue, b6 A117V (selected with WLL), became sensitized to TDI-4258. Hypersensitivity to WHZ-04 and TDI-4258 was also observed in all WLW-selected b2 mutant lines (Figures 2, S1E, and S1F; Tables S1 and S2).
We also tested two modified peptides, compounds 4 and 6 ( Figure 1), which were previously identified as high-affinity, non-covalent inhibitors of the Plasmodium proteasome, targeting the b5 subunit. 20 For these assays, we used three representative mutant lines harboring mutations in b2, b5, or b6, as restricted compound availability precluded additional testing. Both modified peptides exhibited identical resistance profiles, with the WLL-selected b5 A20S line displaying the highest levels of resistance, followed by the b6 A117V line ( Figure 2). Similar to other compounds that primarily inhibit the b5 subunit (e.g., the epoxyketones and AsnEDA compounds), the WLW-selected b2 C31Y mutant line was the most susceptible to both peptide inhibitors (Figures S3D and S3E; Table S1).
Finally, we tested two commercially available agents designed to target the human proteasome, namely the boronate inhibitor bortezomib and the epoxyketone inhibitor epoxomicin ( Figure 1). These inhibitors exhibited moderate or potent activity, respectively, against P. falciparum parasites, consistent with prior studies. 12,13,42 The b5 M45I mutation, selected with MPI-12 (another boronate), conferred the highest level of resistance to bortezomib (Figures 2 and S3F; Tables S1 and S2). The WLL-selected b6 A117D mutation also resulted in increased bortezomib IC 50 values. For epoxomicin, only the b6 S208L mutation resulted in a significant (3-fold) IC 50 shift relative to the corresponding WT line (Figures 2 and S3G; Tables S1 and S2). These results suggest that epoxomicin is minimally affected by mutations that confer resistance to Plasmodium-selective proteasome inhibitors.
Although all of the compounds tested herein inhibit the catalytic b subunits of the 20S proteasome core particle, we have previously shown that mutations in the 19S regulatory particle of the 26S P. falciparum proteasome can also mediate resistance to the vinyl sulfone WLW. 29 We tested whether three WLW-selected 19S mutants, namely RPT4 E380* (premature stop codon), RPT5 G319S, and RPN6 E266K, could mediate cross resistance to any of the other classes of Plasmodium proteasome inhibitors. Consistent with our previous study, 29 none of the three 19S mutations resulted in significant increases in WLL IC 50 values, whereas all three mutations yielded 2-fold lower IC 50 values for the related vinyl sulfone EY 4-78 (Figures S4A-S4C;  Table S3). Small (<2-fold) increases in IC 50 values were also observed for the epoxyketone compounds J-50, J-71, J-78, and J-80 ( Figures S4D-S4G). All three 19S mutations sensitized parasites to the AsnEDA compounds WHZ-04 and TDI-4258 ( Figures S4H and S4I). Conversely, these mutants tended to be more resistant more sensitive  Figure S1 and Table S1. Untransformed fold changes are listed in Table S2.  less sensitive to bortezomib and epoxomicin ( Figures S4J and  S4K). These results suggest that conformational changes imposed by mutations in the 19S regulatory particles may in some cases modulate parasite susceptibility to b subunit inhibitors.
Genetic engineering of inhibitor-selected proteasome mutations reveals that mutations are sufficient to drive resistance To validate the role of drug-selected proteasome b subunit mutations in conferring resistance irrespective of the parasite background, we developed a CRISPR/Cas9 system to edit select b5 mutations, namely A20S (selected for resistance to WLL), A20V (MMV1579506-selected), and M45I (MPI-12selected), into Dd2 parasites ( Figure S5A). Gene-edited parasites were then tested in a new set of 72 h dose-response assays, with the original drug-selected lines harboring the same mutations and their respective parental lines included as controls. Lines were tested against WLL (a covalent, irreversible vinyl sulfone), J-80 (a covalent, irreversible epoxyketone), and TDI-8304 (a non-covalent, reversible macrocyclic peptide). The latter was recently identified as a pharmacologically superior alternative to the AsnEDA TDI-4258. 27 IC 50 values based on dose-response assays showed that the gene-edited lines phenocopied the original drug-pressured lines across each class, confirming that the b5 mutations tested (A20S, A20V, and M45I) were causal for resistance on different genetic backgrounds. For WLL, the b5 A20S edited and selected lines both yielded an 2.5-fold increase in the WLL IC 50 , whereas no increases were observed with the MPI-12-selected M45I mutation and the MMV1579506-selected A20V mutation in either the edited or the drug-pressured lines ( Figures S5B and S2D; Table S4). For J-80 and TDI-8304, all three mutations afforded moderate to high-grade resistance in both the edited and the selected lines ( Figures S5C, S5D, S2E, and S2F; Table S4).
Conditional knockdowns of the b2 or b5 proteasome subunits sensitize parasites to Plasmodium-selective proteasome inhibitors To further validate the Plasmodium proteasome as the target of our different classes of inhibitors, we used CRISPR/Cas9 to engineer conditional knockdown (cKD) parasites in an NF54 Cas9-expressing parasite line (denoted NF54 pCRISPR ). b5 or b2 expression levels were regulated via the TetR-DOZI system (Figure 3A). 50 Basal expression levels were maintained by culturing parasites in the presence of 500 nM anhydrotetracycline (aTc). Medium or low expression levels were obtained for b5 cKD parasites by culturing in 20 or 10 nM aTc, respectively, and for b2 cKD parasites by culturing in 20 or 15 nM aTc, as these concentrations were found to reduce protein expression levels while retaining sufficient parasite growth. Western blot analysis of parasites harvested after 72 h validated protein-level knockdown of both subunits in the absence of aTc ( Figures S6A and S6B). We also observed a significant growth defect in parasites cultured without aTc, consistent with the essentiality of these proteasome subunits ( Figures 3B and 3C).
In the b2 cKD line, decreased levels of b2 were associated with decreased IC 50 values for all proteasome inhibitors tested. At 15 nM aTc, we observed 2to 4-fold lower IC 50 values for WLL (which targets b2 and b5), as well as for EY 4-78, J-71, J-80, and TDI-8304 (which primarily target b5). Lower IC 50 values were also observed at 20 nM aTc ( Figure 3D; Table S5). Control parasites cultured at 50 nM aTc, which allows for normal b5 expression, provided the reference values (we note that all of these compounds were more potent against NF54 parasites than the Cam3.II, Dd2, and V1/S lines tested above). Control assays with chloroquine showed no significant IC 50 changes at the same aTc concentrations.
In the b5 cKD line, we also observed 2-fold lower IC 50 values for all inhibitors tested when cKD parasites were cultured at 10 nM aTc relative to parasites cultured at 50 nM ( Figure 3E). No significant decreases were observed at 20 nM aTc. Chloroquine again showed no significant differences in IC 50 values across aTc concentrations. Thus, although the compounds tested all selected for mutations in b5 and not b2, reduced expression levels of either b2 or b5 led to increased compound sensitivity, potentially because of a negative impact on proteasome complex assembly arising from lowered expression of either individual subunit.
Irreversible Plasmodium-selective proteasome inhibitors display lower rates of in vitro resistance Recent studies have identified resistance liabilities for several antimalarial compounds entering preclinical and human clinical trials, highlighting the need to identify compounds with low resistance risks early in the drug development process. 51 Here, we used in vitro selection experiments with one or two representative compounds from the previously profiled classes of Plasmodiumselective inhibitors, including WLL, TDI-8304, J-71, and J-80, to directly compare resistance risks across chemotypes. To determine the minimum inoculum of resistance (MIR), we exposed WT Dd2 (clone B2) parasites to 3 3 IC 50 concentrations of each compound throughout the selection process (single-step selection). Starting inocula were four wells at 2.5 3 10 6 parasites and three wells at 3 3 10 7 parasites (covering the range from 2.5 3 10 6 to a total of 1 3 10 8 ). Selections were maintained for 60 days or until recrudescence, and recrudescent parasites were cloned by limiting dilution (Figure 4A). For compounds that did not yield resistant parasites at these starting inocula, we performed an additional round of selections with three flasks, each with 3 3  Table S5). Statistical significance was calculated using unpaired t tests with Welch's correction, comparing parasites cultured at the permissive concentration of aTc (50 nM) with parasites cultured under knockdown conditions (10-20 nM aTc); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant.   Table S6). Fold changes are listed in Table S7. Statistical significance was calculated using unpaired t tests with Welch's correction, comparing selected lines to the Dd2 parent; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant.
We next performed resistance selections with the non-covalent reversible inhibitor TDI-8304. This compound yielded resistant parasites with an MIR of 3 3 10 7 (Table 2). Although this compound primarily inhibits b5, TDI-8304 selected for mutations in the b6 subunit, namely N151Y and S157L, which occur in residues that occupy the b5/b6 interface. Strikingly, the b6 S157L mutant line was 2,600-fold more resistant to TDI-8304 than its WT parent, while the b6 N151Y mutant yielded only a moderate (2-fold) increase in IC 50 (Table S7). When tested against J-71, b6 S157L parasites were 2-fold more resistant, whereas b6 N151Y parasites were marginally more sensitive compared with the WT parental line. b6 S157L parasites were 5-fold more resistant to J-80, whereas the b6 N151Y mutation resulted in no change to the J-80 IC 50 (Figures 4B and 4C; Table S7).
For WLL, we were unable to generate in vitro resistance with a total inoculum of up to 10 9 parasites. This finding was consistent with our previously published results in which we obtained lowgrade resistance to the vinyl sulfones WLL and WLW only with very large starting inocula of 2 3 10 9 parasites. 29 We then tested the J-71, J-80, and TDI-8304-selected lines against WLL and its optimized derivative EY 4-78. For WLL, all newly selected b5 or b6 mutations either resulted in no change in IC 50 or yielded a 2-fold increase in susceptibility ( Figure 4E; Table S7). Interestingly, when tested against EY 4-78, the b6 S157L and A117D mutations (selected with TDI-8304 and WLL, respectively) yielded modest (7-and 2-fold) increases in IC 50 values, respectively ( Figures 4F and S3). In contrast, the b6 N151Y line was 2-fold more sensitive to EY 4-78. The b5 M45V and M45I yielded 5-fold higher EY 4-78 IC 50 values, whereas no IC 50 change was observed with the b5 M45R mutant.

Molecular modeling of inhibitor-selected Plasmodium proteasome mutants
We next used modeling to investigate the molecular basis for the resistance of Plasmodium proteasome mutants to their selection compounds. The modeling focused on mutations on the b5 M45, A20, and A50 residues, which were mapped onto the Plasmodium 20S proteasome structure ( Figure 5A). The b5 M45 and A20 side chains are solvent exposed and directly face the b5 P1 binding pocket, suggesting that mutations on these residues could result in changes to compound binding properties without any significant protein conformational changes. However, the b5 A50 side chain is buried, and any mutations on this residue are more likely to lead to local conformational rearrangements associated with changes in intramolecular interactions, affecting the adjacent b5 P1 binding pocket.
Molecular dynamics algorithms are usually used for molecular modeling studies. However, such algorithms are computationally demanding and optimized for studies of small proteins, and thus are not suitable for modeling the full 20S proteasome complex. Previous modeling of Plasmodium proteasome b subunit mutations could be performed only by limiting the models to the two b subunits forming the ligand binding sites. 29 Although informative, this required very careful supervision, as the lack of constraints imposed by the full protein-protein interactions that maintain the 20S proteasome assembly can easily lead to unrealistic model distortions. Here, we used the existing cryo-EM-derived structure of the WT Plasmodium 20S proteasome 21,29 to create structural models of mutant complexes. These models (Figures 5A and 5B) clearly show that the inhibitor-selected b5 M45I, M45R, M45V, A20S, A20V, and A50V mutations all result in spatial contraction of the b5 P1 binding pocket that compromises the binding of each of the selection compounds. Resistance to these compounds can therefore be attributed primarily to steric constraints imposed by the

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Article proteasome mutations. Steric constraints were also associated with compounds for which cross resistance was observed, while sensitization was associated with changes in the electrostatic potential of protein surfaces lining the S1 binding pocket of the mutant proteasomes ( Figure 5B).

DISCUSSION
Combating ART-resistant P. falciparum is a key priority of malaria control and elimination efforts. Proteasome inhibitors have multistage antiplasmodial activity and display synergy with Locations of the b5 A20, M45, and A50 residues (shown as sticks with yellow backbone) in the structure of the wild-type Plasmodium proteasome (shown as cartoon), with fitted WLL (cartoon with gray backbone). 21,29 The table inset shows IC 50 fold shifts for inhibitors tested against proteasome mutant lines compared with the wild-type parental line. Data for the b5 M45I line were taken from Xie et al. 25 (B) Effects of the M45I, M45R, M45V, A20S, A20SV, and A50V mutations on the Plasmodium b5 P1 binding pocket. To facilitate comparison, the b5 P1 binding pocket of the wild-type proteasome (boxed in green) is shown in the same orientation as each of the selection mutants (cyan boxes). For all mutations, the proteasome models indicate that resistance to the selection compounds is primarily mediated by steric constraints that limit their access to the P1 binding site. Examples of sensitization (yellow boxes) and cross resistance (magenta boxes) are also shown. Protein models are represented as Van der Waals surfaces colored by electrostatic potential, overlaid with inhibitors. Article ART, making them attractive candidates for further drug development. Here we characterized a chemically diverse panel of Plasmodium-selective inhibitors, focusing on their resistance properties as a means to prioritize future lead optimization efforts. For these studies, we profiled a panel of covalent and noncovalent inhibitors, including vinyl sulfones, epoxyketones, and boronates in the former category, and AsnEDAs and reversible peptide inhibitors in the latter. By testing these inhibitors against mutant proteasome lines, we identified chemotype-specific mutations whose resistance profiles varied broadly between chemical classes, including instances of collateral sensitivity (Table 3). Overall, the smallest IC 50 shifts across our panel of mutant lines were observed with the vinyl sulfone inhibitors WLL, WLW, and EY 4-78 (up to 3-, 8-, and 2-fold, respectively). For these compounds, no mutations were found that caused IC 50 increases as high as those that earlier proved problematic (30-fold or higher) in human clinical trials with inhibitors targeting dihydroorotate dehydrogenase (DHODH) or the sodium-dependent ATPase PfATP4. 30,32 Unlike other classes of proteasome inhibitors, WLL and WLW exhibited compound-rather than class-specific patterns of resistance. This is most likely attributable to the fact that WLL simultaneously targets the b2 and b5 subunits of the Pf20S proteasome, 21 while WLW primarily targets b2, and all other inhibitors tested herein primarily target b5. Consistent with prior reports, hypersensitization (or collateral sensitivity) to several compounds, including the dual-subunit targeting compound WLL, was mediated by WLW-selected mutations in the 20S b2 subunit. These data suggest that pairing inhibitors with specificity toward different proteasome subunits could serve as an effective tool to mitigate the potential emergence of resistance.
cKD of the b2 and b5 20S subunits sensitized parasites to representative compounds from all three classes of inhibitors examined, but not the control drug chloroquine. For b2, this result was likely the effect of a stoichiometric impact on proteasome assembly rather than direct targeting of this subunit by any of the compounds tested. Indeed, the effect of the b2 cKD was greater than that of the b5 knockdown, likely due to the increased sensitivity of parasites to reduced levels of b2 subunit expression.
MIR selections identified the vinyl sulfone WLL as a particularly refractory inhibitor, with no recrudescence observed at inocula up to 10 9 asexual blood-stage parasites. These data substantiate our prior observation that resistance to vinyl sulfones is relatively difficult to achieve, requiring upwards of 2 3 10 9 parasites. 29 This compares very favorably with new antimalarials recently evaluated in patient exploratory trials, whose MIR values often range from 10 6 to 10 9 parasites, with the lower values associated with increased risk of treatment failure because of readily acquired resistance. 30,32,51 We suspect that the low risk of resistance to WLL is associated with its binding to both the b2 and the b5 subunits. 21 By comparison, selections with the epoxyketones J-71 and J-80 and the macrocyclic peptide TDI-8304 yielded MIR values ranging from 3 3 10 7 (for TDI-8304) to 1 3 10 8 and 3 3 10 8 for J-71 and J-80, respectively. Resistance to these compounds was mediated by mutations in the b5 and b6 subunits. Interestingly, two of these mutations (M45V and M45R) occurred at a residue earlier found to mutate to M45I in response to selection Table 3.

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Article with the potent boronate MPI-12. 25 Our profiling of this M45I mutant line demonstrated that it mediates moderate resistance to several epoxyketone and AsnEDA inhibitors and lower-level cross resistance to the vinyl sulfone inhibitor EY 4-78. WLL and WLW did not lose potency against this mutant line. M45, located at the b5 S1 binding pocket, determines the interactions with the P1 residue of substrates and inhibitors. This residue's methionine side chain is flexible and can project inward to accommodate the small, hydrophobic P1 moiety of some inhibitors, or outward for bulkier substrates. Mutation to an isoleucine or valine introduces larger, hydrophobic side chains that we predict would significantly interfere with binding of inhibitors with large P1 moieties. Modeling of compound-selected mutants of the Plasmodium 20S proteasome strongly supports the notion that resistance to the compounds tested results from mutationimposed steric constraints at the b5 P1 binding pocket. Our data illustrate that resistance profiles can differ even for compounds with similar binding modes, for example, vinyl sulfones and epoxyketones. Both are irreversible, covalent inhibitors, but the vinyl sulfones were markedly less prone to resistance compared to the epoxyketones. This may be due to the fact that some electrophiles are more sensitive to their positioning in the active site relative to the nucleophile, which in the case of the epoxyketones may make them more susceptible to point mutations that alter binding within the active site pockets and affect their ability to form covalent adducts. While our studies suggest that the vinyl sulfones have the most optimal properties for avoiding resistance, their resistance properties are highly compound-specific. Thus, it may be possible to tune the resistance properties of compounds in each of the different classes of inhibitors to minimize the liabilities for resistance generation.
Reversible binding inhibitors (i.e., AsnEDAs and TDI-8304) showed the highest IC 50 shifts (up to 20-to 30-fold) when tested against proteasome mutant lines and were the most prone to acquiring resistance in vitro. This is likely due to the fact that reversible binding compounds reach an equilibrium of bound and unbound states that depends on the K m values for binding. For these compounds, single point mutations can interrupt optimal binding. In the case of covalent compounds, when a similar drop in binding energy occurs, the compounds will bind less efficiently to the active site but will still eventually become covalently bound in place. Over time, covalent inhibitors can continue to block proteasome activity, making generation of resistance more difficult. Any highly significant changes to the proteasome that would effectively prevent inhibitor binding would also likely have an impact on the normal proteolytic function of the proteasome. Thus, our data provide additional support for the use of covalent inhibitors of the Plasmodium proteasome to suppress resistance mechanisms. We note that covalent irreversible inhibitors that are selective for parasite proteasomes have the caveat that any off-target binding to host proteasomes or other host proteases can carry an increased risk of toxicity compared with other types of inhibitors, requiring additional scrutiny during any further drug development efforts. 52,53 For P. falciparum, we note that none of the proteasome mutations selected herein or previously obtained (Tables 2 and S2) were found recently in a sample of 750 Ugandan isolates with sequenced b2 and b5 genes. 54 That study identified a naturally occurring b2 S214F mutation that was associated with a 3and 5-fold higher IC 50 for the peptide boronates MMV1579506 and MPI-12 (also known as MMV1794229), respectively. The vinyl sulfones WLL and WLW as well as the AsnEDA TDI-4258 and the macrocyclic peptide TDI-8304 all retained full activity against the Ugandan isolates tested.
Limitations of the study One limitation of our study is that we profiled only four compounds in the MIR studies, because of the quantity of work required. Additional data would make for a more comprehensive assessment. Another limitation is that our structural modeling was restricted to a subset of b5 mutations and compounds. Additional modeling would provide more insight into the structural basis of resistance and collateral sensitivity. We also did not biochemically profile our compounds against enriched preparations of P. falciparum versus human proteasomes or use activity-based probe profiling to quantify the impact of 20S subunit mutations on inhibitor binding. 22,29 Other crucial factors in drug development remain to be addressed before any Plasmodium-selective proteasome inhibitors can advance to human clinical trials. This includes generating orally bioavailable inhibitors, which in general is challenging for any peptide-based drug. Ultimately, the ideal candidate compound will likely combine features of several of the classes of inhibitors discussed herein while maintaining their activity across multiple parasite life-cycle stages and their unique and established property of synergizing with ART.

SIGNIFICANCE
Malaria's impact on intertropical regions is unrelenting, with an estimated 619,000 deaths in African children below 5 years of age in 2021. Effective treatment with first-line artemisinin-based combination therapies is threatened by artemisinin-resistant parasites that are prevalent in Southeast Asia and are spreading rapidly across eastern Africa. P. falciparum-specific proteasome inhibitors are important assets in the pipeline for new antimalarial drugs as they display the ability to synergize with artemisinin derivatives, including against artemisinin-resistant parasites. These inhibitors bind the catalytic subunits of the proteasome, thereby preventing this multi-subunit complex from reducing artemisinin-induced proteotoxic stress by degrading damaged proteins. Here, we tested whether representatives of the leading chemical classes of Plasmodiumselective proteasome inhibitors differed in their propensity to select for drug-resistant parasites. These compounds differ in their chemical structures and modes of binding. We also assessed the degree to which mutations in the b2 or b5 subunit or proteasome accessory proteins mediated resistance and examined cross-resistance patterns. Our results identify the tripeptide vinyl sulfone WLL as having the most favorable profile, exhibiting a low risk of selecting for resistance and sustained potency against a panel of proteasome mutants. We attribute this feature to the covalent nature of this inhibitor and its irreversible dual binding of the b2 and b5 subunits. Conditional knockdown parasites confirmed compound selectivity for the 20S proteasome. We also identified proteasome mutations that resulted in collateral hypersensitivity, meaning that resistance to one inhibitor caused increased parasite susceptibility to another, creating a potential for resistance-refractory inhibitor combinations. Molecular modeling identified steric constraints in the mutated b5 P1 binding pockets that could reduce drug binding and account for parasite resistance. Our data provide compelling justification for further advancement of proteasome inhibitors with the goal of developing synergistic drug partners to treat artemisinin-resistant malaria.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

DECLARATION OF INTERESTS
The following authors declare the following financial interests: A.E.G. was a Takeda employee; M.D. was an MMV employee; and G. Lin, W. Zhan

Materials availability
Please note that amounts of experimental compounds may be restricted and might require resynthesis. Chemical structures for the compounds used in these studies are shown in Figures 1 and S2A.
Data and code availability d All datasets generated during this study are provided in separate spreadsheets as part of Tables S1-S7. d No code was generated. d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS
Asexual blood-stage parasites were cultured at 3% hematocrit in RPMI-1640 medium supplemented with 50 mM hypoxanthine, 2.1 g/L NaHCO 3 , 2 mM L-glutamine, 25 mM HEPES, 0.5% (w/v) AlbuMAXII (Invitrogen) and 10 mg/mL gentamicin. Parasites were maintained at 37 C in modular incubator chambers supplied with a 5% CO 2 /5% O 2 /90% N 2 gas mixture. Resistance selection and gene editing studies were performed using the Dd2-B2 clone, 56 referred to herein as Dd2. De-identified human erythrocytes were sourced ethically from the Interstate Blood Bank (Memphis, TN) from anonymized blood donors, and their research use for cell culture was in accordance with terms of informed consent under a protocol approved by the Columbia University Medical Center Institutional Review Board, which designated this as not human subjects research.

REAGENT or RESOURCE SOURCE IDENTIFIER
Oligonucleotides See

H NMR for MMV1579506
In vitro drug susceptibility assays IC 50 values for inhibitors against proteasome WT and mutant lines were determined by exposing parasites to serial dilutions of each compound in dose-response assays using asexual blood stage parasites. Compounds were tested in duplicate in 96-well plates, with the final volume per well equal to 200 mL. Parasites were seeded at 0.2% parasitemia and 1% hematocrit. After 72 h, parasites were stained with 13SYBR Green and 100 nM MitoTracker Deep Red (ThermoFisher) and parasite viability was measured on an iQue Plus flow cytometer. IC 50 values were derived by nonlinear regression (GraphPad Prism, version 9).